Expression of pyruvate carboxylase mRNA variants in liver of dairy cattle at calving

Abstract

Background: Bovine liver expresses six pyruvate carboxylase (PC) transcript variants, bPC5'A, bPC5'B, bPC5'C, bPC5'D, bPC5'E, and bPC5'F, which only differ at the 5' untranslated region (UTR) and contain a common coding region. The objective of this experiment was to determine the profile and abundance of PC transcripts in bovine liver at calving.

Methodology/principal findings: A ribonuclease protection assay (RPA) protocol was developed to simplify analysis of these variants and investigate the changes in abundance of each 5' UTR transcript relative to total PC mRNA. Liver biopsy samples collected from seven cows on +1 d relative to calving were analyzed by RPA to determine the profile in PC 5' UTR variants. Results show that all six bovine PC 5' UTR variants are detected at calving. Data indicate that bovine PC 5' UTR variant A is the most abundant, variants C and E are least abundant and expression of variants B, D and F is intermediate at calving.

Conclusions: This manuscript describes a simplified RPA method that quantifies the abundance of six PC transcripts by using two riboprobes. The lack of uniformity in the pattern of PC 5' UTR variants at calving suggests an additional complexity for control of bovine PC mRNA expression at calving that may be the result of transcriptional controls, variation in mRNA processing, or a combination of these processes.

Figure 1. Construction of truncated RPA probes for bovine PC 5′ UTR variant analysis.

Plasmid ABCEF containing all of the elements for bovine PC 5′ UTR was truncated by removing the 96 bp segment near the start of the PC coding sequence to form clone ABCEF_t (Panel A). Clone BCDE containing elements unique to bovine PC 5′UTRs B, C, D, and E was truncated by removing the 96 bp segment near the PC coding sequence to form clone BCDE_t (Panel B). The size of the UTR fragments of the parent and truncated clones is indicated within the shaded and patterned area. In both cases the segment removed included a portion of the 3′ end of the UTR and a portion of the 5′ end of the coding sequence. The coding region is indicated as CR. The adaptor for ligation of BclI and BSU36I digestions is shown in Panel C. The highlighted sequence represents the adaptor and plain text represents the sequence of the 5′ overhang end obtained from BclI and BSU36I digestion of plasmids ABCEF_t and BCDE_t.

Figure 2. Hybridization pattern of riboprobes ABCEF_t and BCDE_t to bovine PC 5′ UTR variants.

Panels A and B describe hybridization pattern of riboprobes ABCEF_t and BCDE_t, respectively. The full sequence of each bovine 5′ UTR variant is represented by the connected series of filled and shaded boxes (that are described in Figure 1) to the left of the variant name. The full length riboprobes are indicated in the top left corner of each panel. Pairing of riboprobe segments with each 5′ UTR variant is indicated below the boxes representing each variant. Upon digestion with RNase H and T1 the sense and antisense paired strands of RNA remaining in solution are indicated for each UTR variant and coding sequence. The sizes of the protected fragments that originate from hybridization with each riboprobe are given on the right. Both probes protect an identical 59 nucleotide fragment of the coding sequence of bovine PC.

Figure 3. Autoradiogram of ribonuclease protection assay for bovine 5′ UTR variants.

Total RNA samples were hybridized in solution with either riboprobe ABCEF_t or BCDE_t and digested with a mixture of RNase A and RNase T1. The protected fragments were separated by electrophoresis through a 5.5% polyacrylamide, 7 M urea gel. The protected fragments were visualized by exposing the dried polyacrylamide gel to Kodak X-Omat film and quantified using Kodak 1D image analysis software (Version 2.0.1). Fragments for transcripts bPC5′A, bPC5′B, bPC5′C, bPC5′D, bPC5′E, and bPC5′F are indicated by the letters A, B, C, D, E, and F respectively. The abbreviations CR, M, UE, and UC represent the coding region, molecular weight markers, undigested probe BCDE_t, and undigested probe ABCEF_t, respectively. Size of molecular weight markers are shown on the left of the gel.

Figure 4. Contribution of PC 5′UTR variants to total PC mRNA on +1 d relative to calving.

RNA samples were subjected to RPA analysis and the abundance of protected fragments was adjusted for the number of uracil residues within each fragment. Abundance of each variant was determined by dividing the uracil-adjusted values for each variant by the uracil-adjusted value of coding region generated from riboprobe ABCEF_t and riboprobe BCDE_t. Means separation was by Duncan's Multiple Range Test and means that are statistically different (P<0.05) are indicated by superscripts (a, b, c, and d) above the bars that differ.